A characteristic structural component of the cell wall of bacteria is peptidoglycan. The peptidoglycan cell wall surrounds the bacterial cell, provides structural support, and shields the bacterial membrane against osmotic rapture. It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues connected with β(1,4)-glycosidic bonds [Boneca et al., 2000] and cross-linked with short polypeptide chains. Bacterial growth, division, colonization, and biofilm formation heavily relies on the ability of the cells to remodel their cell wall. Therefore, many antibiotics, such as penicillin, target the biosynthesis of the cell wall. Due to the increasing resistance of bacteria to antibiotics, it is important to find alternative targets for the treatment of bacterial infections, e.g. enzymes involved in the cell wall degradation. Cell wall degradation is performed by peptidoglycan hydrolases, also known as autolysins [Smith et al, 2000]. Among them are glycosidases which hydrolyze glycosidic bonds in NAM-NAG polymers. There exists two major types of glycosidases, N-acetylglucosaminidases and N-acetylmuramidases (lysozyme-like proteins), which cleave the β(1,4)-glycosidic bonds between NAG-NAM, and NAM-NAG residues, respectively [Vollmer et al., 2008]. As glucosaminidases and muramidases act on the same substrate, it is generally useful to gain insight into their biochemical properties and reveal features which enable them to perform the two distinct functions.
Staphylococci represent a large group of bacteria which inhabit humans and can cause severe infections to people with weak or compromised immune system. Among them is Staphylococcus aureus (S. aureus) which is responsible for most of hospital acquired infections [Vincent et al., 2009]. S. aureus is a Gram-positive bacterial pathogen that is responsible for severe medical conditions in humans, including bacteremia, endocarditis, metastatic infections, sepsis and toxic shock syndrome [Lowy, 1998], osteomyelitis [Varonne et al., 2011]. S. aureus possesses the capability to form biofilms during the progress of infection which is of advantage of the bacteria. Biofilms offer protection of the cells against antibiotics and the host immune response which leads to the development of a long and persisting chronic disease [Archer et al. 2011]. Since S. aureus was the first human pathogen treated with antibiotics, strains resistant to the antibiotics already emerged a while ago, e.g. resistance against β-lactame antibiotics as penicillin, methicillin (Methicillin Resistant S. Aureus—MRSA) or vancomycin (Vancomycin Resistant S. Aureus—VRSA) [Hiramatsu et al., 1997], [Zetola et al., 2005], [Dantes et al., 2013], [Gardete and Tomasz, 2014].
The genome of a S. aureus strain, which is resistant to vancomycin (Mu50), encodes five putative GH73 family (Glycoside Hydrolase Family 73) members. Four of them, SAV2307, SAV1052, SAV1775, and SAV2644, are widely distributed through the genomes of S. aureus strains. In addition, the genome of S. aureus Mu50 strain encodes SAV0909, which was inserted into the genome through the integration of Bacteriophage phi mu1.The best studied among them is (SAV1052) Major Bifunctional Autolysin (AtlA) [Oshida et al., 1995]. The AtlA deletion mutants form large cell clusters and are biofilm negative [Heilmann et al. 1997, Biswas et al. 2006, Sugai et al. 1995]. The AtlA gene encodes two activities: amidase and glucosaminidase encoded at the N-terminal and C-terminal regions of the sequence, respectively. The amidase activity of AtlA was confirmed and analyzed by structural studies of a homologous enzyme from Staphylococcus epidermis [Zoll et al., 2010] and later S. aureus [Buttner et al, 2014]. However, the glucosaminidases and their role in biofilm formation remained unexplored.
The crystal structures of two GH73 members from Lysteria monocytogenes [Bublits et al., 2009] and of the C-terminal domain of the flagellar protein FlgJ from Sphingomonas sp. [Hasihimoto et al., 2011] and recently endo-N-acetylglucosaminidase from Streptococcus pneumoniae (LytB SP) [Bai et al., 2014] were determined. While the first two proteins exhibit only remote sequence homology to S. aureus, the last one is in part closely related, yet distinct from GH73 family of glucosaminidases from S. aureus. This indicates that members of the GH73 family are sequentially divergent and constitute distinct subfamilies.